This application addresses broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic 01-CA-102, The Role of Nutrition in Cancer Biology. Our long term goals are to characterize the role of genomic stability in dietary prevention of cancer and to develop an intermediate bioassay of DNA damage for testing efficacy of dietary agents. These studies will provide proof of principle of this concept and establish the bioassay through study of vitamin D and genomic stability. This project specifically targets the Challenges "Prevention" and "Role of Nutrition in Cancer Biology" in the following ways: 1. Focus on normal mammary cells and cancer prevention. My lab has extensively studied the effects of vitamin D on breast cancer cells with the idea of targeting the vitamin D receptor (VDR) for cancer therapy. Unfortunately, in clinical trials, patients had late stage cancer and exhibited minimal improvement with vitamin D based therapies. Similar results have been reported with many other nutrients and nutrient derivatives (ie, vitamin A, vitamin E, etc). Thus, we began studying the effects of vitamin D on normal mammary gland with the idea that vitamin D would be effective in prevention of cancer. Indeed, we showed that VDR null mice display enhanced sensitivity to chemical and oncogene induced carcinogenesis. This proposal directly targets the Broad Challenge Area of Prevention with the plan to comprehensively study how vitamin D treatment impacts on DNA damage response pathways in normal human and mouse mammary cells in vitro and in vivo. 2. Testing dietary vitamin D supplementation in vivo. Dietary requirements for vitamin D were originally set for prevention of the bone disease rickets. At the time, a role for vitamin D in cancer prevention was not recognized. Thus, we know how much vitamin D is required for optimal bone growth, however, we do not know how much vitamin D is required for optimal health of other tissues that express the VDR such as the breast. One innovation of our approach is that we will increase dietary vitamin D 5, 10 and 20 fold above the "standard rodent requirement". This is based on the fact that we just don't know how much is enough (we do know from our previous studies on calcium metabolism that these levels are not toxic). 3. Development of an intermediate bioassay for testing effects of chemopreventive agents on the DNA damage response. Although cell based studies have begun to demonstrate effects of nutrients on genomic stability, extension of in vitro data to a more complex tissue environment that mimics cancer development in vivo is obviously a challenge. With respect to monitoring DNA damage resulting from double strand breaks (DSB), in vivo studies are especially difficult because genotoxic agents induce DSBs at random sites in multiple tissues, and it is the error-prone repair of these breaks that cause mutations throughout the genome. The initial damage to the DNA is no longer identifiable once cancer eventually develops. We therefore propose to validate a bioassay using mammary gland in organ culture exposed to genotoxic agents (we will use radiation and doxorubicin, but this assay could be adapted for other genotoxic agents of interest). This will allow exact control over exposure to genotoxic agents within the mammary gland, and monitoring of the immediate damage (DSBs). In addition, we believe that a bioassay that utilizes intact tissue will more closely model the in vivo biology relevant to cancer prevention. A major strength of our design will be the ability to manipulate the diets of mice prior to removal of the glands for culture and thus correlation can be made between dietary/serum levels of nutrients and effectiveness in the bioassay. Our aim for this two year challenge grant is to provide proof of principle by studying the effects of supplemental dietary vitamin D with this approach. If validated, this bioassay could prove useful as an intermediate approach for screening dietary agents that optimize DNA damage response pathways. Alternatively, of course, one could use knockout or transgenic mice as donors to probe specific gene-nutrient interactions in modulation of DNA damage response pathways in the mammary gland. 4. Initiation of a prevention genomic database. Another attractive aspect of our approach is that one can collect multiple glands from the same animal and simultaneously compare effectiveness of chemopreventive agents in the bioassay with genomic or proteomic profiles. In contrast to cell culture studies, the bioassay will allow profiling of gene expression changes in the context of all mammary cell populations (epithelial, stromal, immune, endothelial, etc). In the long term, a database of genomic profiles induced by preventive agents effective in this bioassay will be developed. Ultimately, this database is expected to generate a common set of biomarkers associated with cancer preventive agents that maintain genomic stability. PUBLIC HEALTH RELEVENCE: People are continuously exposed to environmental agents that can cause genetic mutations and increase the risk of cancer development. Fortunately, healthy tissues can repair the majority of these mutations, some cells do eventually accumulate enough damage over time that they become cancerous. Based on this paradigm of age-related accumulation of damage, it stands to reason that dietary factors might delay cancer development by maximizing tissue repair processes. In this project we will develop methods to study damage and repair processes in mammary gland, and will perform initial experiments to test whether vitamin D acts to prevent breast cancer via enhancement of cellular protective responses.